Her hypothesis was drawn out as a cartoon on the whiteboard in her lab:

Available Reagents
Using a yeast one-hybrid screen , she has isolated a mouse cDNA clone that encodes a DNA binding protein with high affinity for the TTCAAT sequence. The sequence of the mouse Igs cDNA clone was used in a GenBank Blast search through the internet to identify a human EST that was later found to encode a portion of the human Igs gene homologue.

With the Igs partial cDNA in hand, she screened a pancreas-specific mouse cDNA library and isolated the full-length Igs coding sequence which she recloned to generate a single cDNA.

Basic Strategies

Specific Aim 1

Development of an in vivo functional assay to analyze Igs activity
In the first set of experiment she cloned the Igs ORF into the eukaryotic expression vector pCMV. She also constructed two Igs reporter genes using the pF-luc vector. In one strategy she cloned in the insulin gene 5' regulatory region to create pF-luc-Inslin, and in another scheme, she inserted four copies of the consensus Igs binding element (TTCAAT) to generate pF-luc-IBx4 .

Two human pancreatic-derived cell lines are available to her in the lab. One is called "PanC" which represents normal pancreatic cells and is known to express both Igs and SLH proteins. The other cell line is called "DiB-TC", and it is known to express SLH proteins but NOT Igs based on Norhtern Blots. DiB-TC was isolated from an insulin-dependent diabetic patient.

Based on the finding that the addition of exogenous Igs to PanC cells did not increase expression of the pF-luc-Insln or pF-luc-IBx4 reporter genes above that of pCMV alone, she proposes that SLH proteins are the limiting factor in the formation of Igs-SLH complexes. Moreover, activation of the same F-luc reporter genes in DiB-TC cells was found to be dependent on the addition of pCMV-Igs plasmid which is consistent with the lack of endogenous Igs protein in these cells.

Specific Aim 2

Functional mapping of Igs using gene expression assays in cultured cells
Now that she has the full-length Igs ORF in hand, and more importantly, a functional assay worked out in DiBC-TC cells that is Igs-dependent, she wants to define Igs seqeunces that are required for transcriptional regulation of the IBS4 and human insulin reporter genes. This was done by constructing in-frame deletion mutants of the Igs coding sequence. With this deletion mutants, she can then test for in vivo activity by the transient transfection assay, and in addition, measure in vitro DNA binding using bacterially expressed protein in combination with the electrophoretic mobility shift assay (EMSA).



Generation of Igs N-terminal and C-terminal deletion mutants using the pBluescript vector.

Based on the data shown above, the scientist predicts that in addition to a central region required for in vitro DNA binding, thera area at least two regions of the Igs protein coding sequence that may function in vivo as activation domains which are required for full activity.

Just for fun, she printed out the molecular structure of a typical transcription factor complex bound to DNA to get an idea of how Igs-SLH interctions may be critical to gene regulation. This example is from the Protein Data Bank and is the molecular structure of Runx-1/cEBP complex.

What is the difference between the yeast Two-hybrid screen and the yeast One-hybrid screen with regard to the research objective (i.e., what do you need as materials for each strategy and what is the outcome)?





What could the scientist do if the pancreatic library she was screening did not contain any clones corresponding to the 5' end of the ORF (the library was made with oligo dT)? Describe two approaches and the advantages/disadvantages of each.



Based on the observation that IBx4 reporter gene worked almost as well as the insulin reporter gene in this assay, what does this suggest about the mechanism of Igs in insulin gene regulation? Which experiment with PanC cells supports this conclusion?

One interpretation of the terminal deletion experiments is that protein domains required for Igs function (SLH interactions) are encoded by the deleted amino acids. What is an alternative explanation that might explain the loss of Igs activity?



Why is it necessary to engineer in the ATG and TAG codons into the coding sequence of the N-terminal and C-terminal deletion mutants,respectively? How would you do this?


What is the most desireable explanation for the "super-shifted" band in the EMSA assay when the Dib-TC protein extract is added to the binding reaction? How would you distinguish between the presence of an Igs-specific and Igs non-specific protein activity in the Dib-TC extract?

Specific Aim 3

Identification of Igs residues that interact with SLH proteins in Dib-TC cells
To test her hypothesis that Igs protein functionally interacts with SLH transcription factors present in Dib-TC cells, she performed the following two experiments;

1) Gain of function tests using the yeast Gal4 DNA binding domain fused to regions of the Igs protein that she suspects interact with SLH proteins.

2) Co-immunoprecipation studies using retroviral vectors containing Flag-tagged Igs deletion mutants. For these experiments, anti-Flag antibodies were used to determine which of the retrovirally-transduced Igs deletion mutants interact in vivo with SLH proteins based on Western blotting of the Flag immunoprecipates with anti-SLH antibodies.

The strategy and results of these two experimental approaches are shown below.

Gain of function strategy confirms that Igs encodes N-terminal and C-terminal activation domains.